Natural killer (NK) cells are able to specifically recognize and lyse certain tumor or virally-infected cells without prior sensitization to targets. The cell surface molecules responsible for the specificity of the NK-target cell interaction are not defined. Recent structural and functional evidence suggests that the cell surface glycoprotein NKR-P1 on rat NK cells may serve as an important receptor in the recognition of target cells. The identification of ligands on target cells which bind to NKR-P1 will help to define the mechanisms underlying the NK cell response. NKR-P1 is a type II integral membrane protein with an extracellular C-type lectin domain. The lectin region is structurally similar to known receptor molecules. Our laboratory has generated a mutant rat NK cell line (derived from RNK- 16) lacking expression of NKR-P1, which is selectively unable to lyse certain target cells derived from C57BL/6 (H-2b) mice. Other target cell lines are killed equally by mutant and wild type RNK-16 cells. These results support the hypothesis that NKR-P1 may be required for recognition and/or lysis of some but not all target cells. My proposed studies will focus on defining the ligand(s) for NKR-P1 on target cells. I have prepared a recombinant soluble chimeric protein composed of the extracellular domain of NKR-P1 fused to the Fc portion of human IgG1 (rNKR-P1/Fc). Utilizing this chimeric protein as a probe for ligand, target cells will be screened by FACS, by cell binding to immobilized chimeric protein, and by antibody-dependent cell mediated cytotoxicity. To demonstrate that target cell interactions with soluble chimeric rNKR-P1/Fc are analogous to cell surface protein interactions, CHO cells, transfected to express high levels of cell surface NKR-P1, will be used to examine specific cell-cell adhesion to targets. Ligand(s) for NKR-P1 will be affinity-purified from appropriate target cells using immobilized rNKR-P1/Fc. Putative ligand(s) will be characterized with regard to size, susceptibility to proteolysis, carbohydrate content, and relation to known cell-surface molecules. These studies will identify ligand(s) for NKR-P1, a proposed receptor on NK cells. Identification of the molecules involved in the specificity of NK-target cell recognition will provide important insight into the role of NK cells in immune regulation and how they might discriminate between tumors and non-neoplastic cells.